Description
Human Cystathionine gamma-lyase (CTH) ELISA Kit
The Human Cystathionine Gamma-Lyase (CTH) ELISA Kit is specifically designed for the sensitive and accurate detection of CTH levels in human serum, plasma, and cell culture supernatants. This kit offers high specificity and precision, ensuring dependable and reproducible results that are crucial for various research applications.CTH is an important enzyme involved in the regulation of sulfur-containing amino acids and hydrogen sulfide production in the body. Dysregulation of CTH has been linked to various diseases, including cardiovascular disorders, neurological conditions, and metabolic disorders.
As such, measuring CTH levels can provide valuable insights into the pathogenesis of these diseases and potential therapeutic interventions.With its reliable performance and ease of use, the Human CTH ELISA Kit is an indispensable tool for researchers studying the role of CTH in health and disease. Get accurate and insightful results with this advanced kit from Assay Genie.
| Product Name: | Human Cystathionine gamma-lyase (CTH) ELISA Kit |
| SKU: | HUEB1762 |
| Size: | 96T |
| Target: | Human Cystathionine gamma-lyase (CTH) |
| Synonyms: | Cysteine-protein sulfhydrase, Gamma-cystathionase |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.78-50ng/mL |
| Sensitivity: | 0.23ng/mL |
| Intra CV: | 6.7% | ||||||||||||||||||||
| Inter CV: | 8.9% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Catalyzes the last step in the trans-sulfuration pathway from methionine to cysteine. Has broad substrate specificity. Converts cystathionine to cysteine, ammonia and 2-oxobutanoate. Converts two cysteine molecules to lanthionine and hydrogen sulfide. Can also accept homocysteine as substrate. Specificity depends on the levels of the endogenous substrates. Generates the endogenous signaling molecule hydrogen sulfide (H2S), and so contributes to the regulation of blood pressure. Acts as a cysteine-protein sulfhydrase by mediating sulfhydration of target proteins: sulfhydration consists of converting -SH groups into -SSH on specific cysteine residues of target proteins such as GAPDH, PTPN1 and NF-kappa-B subunit RELA, thereby regulating their function. |
| Uniprot: | P32929 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Cystathionine gamma-lyase |
| Sub Unit: | Homotetramer. Interacts with CALM in a calcium-dependent manner. |
| Research Area: | Cell Biology |
| Subcellular Location: | Cytoplasm |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | CTH: Catalyzes the last step in the trans-sulfuration pathway from methionine to cysteine. Has broad substrate specificity. Converts cystathionine to cysteine, ammonia and 2-oxobutanoate. Converts two cysteine molecules to lanthionine and hydrogen sulfide. Can also accept homocysteine as substrate. Specificity depends on the levels of the endogenous substrates. Generates the endogenous signaling molecule hydrogen sulfide (H2S), and so contributes to the regulation of blood pressure. Acts as a cysteine-protein sulfhydrase by mediating sulfhydration of target proteins: sulfhydration consists of converting -SH groups into -SSH on specific cysteine residues of target proteins such as GAPDH, PTPN1 and NF-kappa-B subunit RELA, thereby regulating their function. Defects in CTH are the cause of cystathioninuria (CSTNU). It is an autosomal recessive phenotype characterized by abnormal accumulation of plasma cystathionine, leading to increased urinary excretion. Belongs to the trans-sulfuration enzymes family. 2 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:EC 4.4.1.1; Amino Acid Metabolism - cysteine and methionine; Lyase; Energy Metabolism - nitrogen; Cell cycle regulation; Amino Acid Metabolism - glycine, serine and threonine; Other Amino Acids Metabolism - selenoamino acid Chromosomal Location of Human Ortholog: 1p31.1 Cellular Component: nucleoplasm; cytoplasm; cytosol; nucleus Molecular Function:calmodulin binding; protein binding; cystathionine gamma-lyase activity; homocysteine desulfhydrase activity; carbon-sulfur lyase activity; pyridoxal phosphate binding Biological Process: cysteine metabolic process; positive regulation of I-kappaB kinase/NF-kappaB cascade; sulfur amino acid metabolic process; unfolded protein response; sulfur amino acid catabolic process; protein-pyridoxal-5-phosphate linkage via peptidyl-N6-pyridoxal phosphate-L-lysine; transsulfuration; cysteine biosynthetic process; activation of NF-kappaB transcription factor; protein homotetramerization Disease: Cystathioninuria |
| NCBI Summary: | This gene encodes a cytoplasmic enzyme in the trans-sulfuration pathway that converts cystathione derived from methionine into cysteine. Glutathione synthesis in the liver is dependent upon the availability of cysteine. Mutations in this gene cause cystathioninuria. Alternative splicing of this gene results in three transcript variants encoding different isoforms. [provided by RefSeq, Jun 2010] |
| UniProt Code: | P32929 |
| NCBI GenInfo Identifier: | 27735163 |
| NCBI Gene ID: | 1491 |
| NCBI Accession: | P32929.3 |
| UniProt Secondary Accession: | P32929,Q53FB3, Q53Y79, Q9H4W7, Q9H4W8, B4E1R2, E9PDV0 |
| UniProt Related Accession: | P32929 |
| Molecular Weight: | 41,260 Da |
| NCBI Full Name: | Cystathionine gamma-lyase |
| NCBI Synonym Full Names: | cystathionine gamma-lyase |
| NCBI Official Symbol: | CTH |
| NCBI Protein Information: | cystathionine gamma-lyase; gamma-cystathionase; homoserine deaminase; cysteine desulfhydrase; homoserine dehydratase; cysteine-protein sulfhydrase; cystathionase (cystathionine gamma-lyase) |
| UniProt Protein Name: | Cystathionine gamma-lyase |
| UniProt Synonym Protein Names: | Cysteine-protein sulfhydrase; Gamma-cystathionase |
| Protein Family: | Cystathionine gamma-lyase |
| UniProt Gene Name: | CTH |
| UniProt Entry Name: | CGL_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
